Genome Transplant Dynamics: non-invasive sequencing-based diagnosis of rejection

描述（由申请人提供）：我们的目标是开发和测试一种新的供体特异性基因组方法，用于实体器官移植后排斥反应和移植物功能障碍的非侵入性早期诊断，并解决两个目标主题领域：1)应用基因组学和其他高通量技术；2)将基础科学发现转化为新的更好的治疗方法。器官移植可以挽救终末期器官衰竭患者的生命，但在许多情况下，移植的器官会被受体排斥，造成危及生命的情况。先前开发移植物排斥反应的非侵入性标志物的尝试主要集中在受体特异性免疫反应上，因此在敏感性和特异性上存在固有的局限性，特别是在区分排斥反应和感染方面。我们的新方法是第一个专注于供体特异性急性排斥标志物的方法。我们将使用高通量下一代测序来监测受体血液中无细胞供体DNA与受体DNA的比例，作为排斥反应的标志。这种方法是可行的，因为器官移植实际上也是一种基因组移植，通过监测供体基因组特有的单核苷酸多态性，可以测量移植器官的相对健康状况。理由/假设：在排斥反应中，供体细胞的凋亡将供体DNA释放到受体循环中；该DNA可以与受体DNA区分，并使用高通量测序技术进行量化；供体DNA水平的动态变化可以预测(a)急性排斥反应；(b)移植物功能障碍；(c)慢性排斥。初步研究：使用心脏移植受者的血液样本，我们发现供体DNA水平在急性排斥发作期间上升，但在没有排斥反应时保持稳定的低水平。目的1：开发一种供体DNA监测方法，用于无创检测同种异体移植排斥反应。我们将开发和测试：(1)下一代DNA测序方法，以执行低成本，无创分析供体DNA负载；(2)最大化区分供体和受体DNA敏感性的生物信息学算法；(3)区分供体和受体的单核苷酸多态性（SNP）面板的敏感性。目的2：评估供体DNA监测对心脏移植后临床事件（急性排斥反应、慢性排斥反应和移植物功能障碍）检测的效用。我们将对110名连续的心脏移植受者进行前瞻性队列研究，在心脏内膜心肌活检（EMB）手术期间和之间收集血液样本（产生1516对EMB/血液），以确定供体：受体DNA比率是否可以在早期阶段和移植物功能障碍发展之前检测排斥反应。目的3：在肺移植中测试这种方法，并确定供体：受体DNA比例是否可以区分排斥反应引起的移植物功能障碍与肺部感染。如果实现，这些特定目标将为实体器官移植后非侵入性早期排斥检测的供体特异性基因组方法的验证研究提供基础。

DESCRIPTION (provided by applicant): Our goal is to develop and test a novel donor-specific, genomic approach to the non-invasive, early diagnosis of rejection and graft dysfunction after solid organ transplantation, and addresses two targeted thematic areas: 1) Applying Genomics and Other High Throughput Technologies; 2) Translating Basic Science Discoveries into New and Better Treatments. Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. Previous attempts to develop a non-invasive marker of graft rejection have focused on recipient-specific immune responses, and thus have inherent limitations in sensitivity and specificity, especially for distinguishing rejection from infection. Our novel approach is the first to focus on a donor-specific marker of acute rejection. We will use high throughput next generation sequencing to monitor the proportion of cell-free donor DNA to recipient DNA, in the recipient's blood stream as a marker of rejection. This approach is enabled by the fact that an organ transplant is also effectively a genome transplant, and by monitoring single nucleotide polymorphisms that are specific to the donor's genome one can measure the relative health of the transplanted organ. RATIONALE/HYPOTHESIS: during rejection, apoptosis of donor cells releases donor DNA into the recipient circulation; this DNA can be distinguished from recipient DNA and quantified using high-throughput sequencing techniques; and dynamic changes in donor DNA levels will predict (a) acute rejection; (b) graft dysfunction; and (c) chronic rejection. PRELIMINARY STUDIES: using banked blood samples from heart transplant recipients, we showed that donor DNA levels rise during episodes of acute rejection but remain at stable, low levels in the absence of rejection. AIM 1: Develop a donor DNA monitoring approach for the non-invasive detection of allograft rejection. We will develop and test: (1) next generation DNA sequencing methods to perform low cost, noninvasive analysis of donor DNA load; (2) bioinformatic algorithms that maximize the sensitivity to discriminate donor and recipient DNA; (3) sensitivity of a defined single nucleotide polymorphism (SNP) panel that differentiates donor from recipient. AIM 2: Evaluate the utility of donor DNA monitoring for detection of clinical events (acute rejection, chronic rejection, and graft dysfunction) after heart transplantation. We will conduct a prospective cohort study of 110 consecutive heart transplant recipients, collecting blood samples during and between endomyocardial biopsy (EMB) procedures (yielding 1,516-paired EMB/blood) to determine whether donor:recipient DNA ratio can detect rejection in its early stages and before the development of graft dysfunction. AIM 3: Test this approach in lung transplantation and determine whether donor:recipient DNA ratio can differentiate between graft dysfunction due to rejection versus pulmonary infection. If achieved, these specific aims will provide the foundation for validation studies of a donor-specific, genome-based approach to non-invasive, early detection of rejection after solid organ transplantation. PUBLIC HEALTH RELEVANCE: Organ transplantation saves the lives of patients with end-stage organ failure, yet in many cases, the transplanted organ is rejected by the recipient, causing a life-threatening situation. We propose to develop and test a new and innovative approach to the non-invasive, early diagnosis of rejection and graft dysfunction after heart and lung transplantation. By simply monitoring the blood of a transplant recipient for level of the donor genome appearing during follow-up after receiving a transplant, we hope to establish a diagnostic tool that has potential to save hundreds of millions of dollars in health care costs annually, and reduce patient morbidity and mortality.